A majority of human cancers originate from epithelial tissue. A common cancer of epithelial origin is nonmelanoma skin cancer (NMSC), including basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), with more than 700,000 new cases diagnosed each year in the United States. Similar cancers are also seen in non-human animals such as domesticated animals and pets, including cats and dogs. BCC is rarely life-threatening because it is slow growing and is mostly localized. Unlike BCC, SCC metastasizes at a rate of 2% to 6% over several years after initial diagnosis. A highly malignant form invades and destroys tissue, and then metastasizes, initially to a regional lymph node before more distant organs such as the lung or brain are affected. SCC is commonly encountered in a number of epithelial tissues, including the oral cavity, esophagus, larynx, bronchi, intestines, colon, genital tract, and skin. Early detection using reliable biomarkers is desired, as are rationally designed drugs for effectively preventing and treating aggressive, metastatic SCC.
As such, there is a need for a good animal model system for studying how metastatic squamous cell carcinoma develops, progresses and can be treated. To date, no such model exists. Classically, tumor cells are injected into the tail vein of either immunocompromised or syngeneic mice. While this assay can suitably model the later metastatic stages, it does not model the early genesis, invasion and angiogenic stages of malignant progression, especially as it relates to complex interactions between tumor and host, especially at the tissue site where the carcinoma originated. Moreover, the role of the immune system in metastatic progression cannot be analyzed when immunocompromised mice are used.
Murine skin model systems are still essential contributors to our understanding of the multi-step nature of chemically-induced carcinogenesis. In the multistage mouse skin carcinogenesis model, biochemical events unique to initiation, promotion, or progression can be studied and related to cancer formation. In that model, the NMSC that is most often induced is squamous cell carcinoma. Although squamous cell carcinoma of mouse skin invades the dermal region, the incidence of malignant metastatic conversion is rare and requires a long latency period of approximately a year.
Several protocols are used to develop mouse skin tumors in laboratory animals. In a common initiation-promotion protocol, mouse skin is treated with an initiating agent (7,12-dimethylbenz[a]anthracene; DMBA) and then with a potent tumor promoter (12-O-tetradecanoylphorbol-13-acetate; TPA). In this protocol, mice develop mostly benign papillomas, more than 90% of which regress after TPA treatment is stopped. Only a small percentage of papillomas progress to invasive, but non-metastatic, SCC. The initiation-promotion protocol has been further modified to enhance the conversion of skin papillomas to carcinomas, yet metastatic potential is not increased.
A major intracellular receptor for TPA is the ubiquitous enzyme protein kinase C (PKC), an important signal transduction pathway component for controlling cell proliferation and tumorigenesis. It has been suggested that PKC activation may play a role in promoting mouse skin tumor formation. However, several groups have demonstrated that repeated applications of TPA depress PKC activity and protein levels. These results indicate that both loss of PKC activity and degradation of PKC could be important for mouse skin tumor promotion by TPA.
On the basis of the structural similarities and cofactor requirements, the eleven known PKC isoforms are grouped into three subfamilies: (1) the conventional PKCs (α, βI, βII, and γ), which depend upon Ca2+, phosphatidylserine (PS), and diacylglycerol (DAG) or TPA; (2) the nPKCs (ε, δ, η, and θ), which require only PS and DAG/TPA; and (3) the a typical PKCs ι/λ and ζ), which retain PS dependence but have no requirement for Ca2+ or DAG/TPA for activation. PKCμ, which is usually classified as a nPKC, is not easily grouped with any of the other isoforms.
The roles of PKCα and PKCδ isoforms in the mouse skin tumor initiation/promotion protocol were assessed in FVB/N transgenic mice expressing an T7-epitope-tagged PKCα (T7-PKCα) or PKCδ (T7-PKCδ) under the control of the human keratinocyte-specific K14 promoter/enhancer. Transgenic expression of T7-PKCα did not affect tumor promotion susceptibility. Transgenic expression of T7-PKCδ in the epidermis (˜8-fold increase) suppressed the formation of both skin papillomas and carcinomas by 70%.
PKCε may play an important role in cellular growth regulation. TPA binds to and activates PKCε. Activated PKCε may be important for the survival of small cell lung carcinoma cell lines in which the catalytic fragment of PKCε is constitutively expressed. Overexpression of PKCε in Rat-6 or NIH-3T3 fibroblasts increases growth rate, anchorage independence, and tumor formation in nude mice. PKCε overexpression also transforms non-tumorigenic rat colonic epithelial cells and suppresses apoptosis of interleukin-3 dependent human myeloid cells induced by removal of interleukin-3.
The role of PKCε in mouse skin tumor promotion and epidermal cell growth and differentiation remains unclear. Treatment of the mouse skin with TPA leads to a general reduction in PKC activity that persists for at least 4 days. Acute TPA treatment decreases PKCβ and η protein levels, but has little or no effect on the levels of PKCα, δ, or ε. PKCα, β, and δ activity levels were reduced after acute or repeated TPA treatments, but PKCε activity was not examined. DMBA/TPA-induced papillomas exhibit decreased cytosolic levels of PKCα and βII protein, but insignificant alterations in the levels of PKCδ, ε, or ζ protein. When cultured mouse skin keratinocytes are induced to differentiate by increasing Ca2+, PKCε, δ, and α translocate to the membrane fraction, suggesting a role for activation of these isoforms in keratinocyte differentiation.
Chronic exposure to UV radiation in sunlight is an important risk factor for non-melanoma epidermal carcinogenesis and for other common recurrent skin injuries such as sunburn and premature cutaneous photoaging. The UV spectrum, having wavelengths between those of visible light and x-rays, is conventionally divided into three major wavelength groups: UVA (315-400 nm), UVB (280-315 nm) and UVC (190-280 nm). UVA and UVB are the most prominent and ubiquitous carcinogenic wavelengths, as ozone in the stratosphere absorbs most of the radiation below 310 nm. The UVA and UVB components are highly genotoxic, but do not penetrate deeper than the skin. The biological effects mediated by UVA and UVB are subtly different.
UV radiation is considered to be a complete carcinogen because it both initiates and promotes carcinogenesis. UVB induces oxidants that directly damage DNA and induce gene mutations, the key initiating genetic events in carcinogenesis. Gene mutations associated with UV-induced skin cancer include TP53, PITCH and ras oncogenes. UV-induced oxidants may also regulate a protein kinase network that controls gene expression associated with skin tumor promotion. UV radiation also potently suppresses the immune system.